ABSTRACT Refractoriness to treatment is a major cause of asthma-related morbidity and mortality. Refractory asthma accounts for the bulk of the direct cost for asthma. There is a general consensus that asthma is heterogeneous and that the same treatment will not work for all. Treatments that failed in general asthma population were found beneficial to a specific endotype of asthma (e.g. anti-IL5 antibody in eosinophilic asthma). For this reason identification of mechanism-based subtypes (endotypes) of asthma and development of mechanism-targeted treatment are of paramount importance. Using flow cytometry-based characterization of bronchoalveolar lavage (BAL) cells we have recently reported the identification of a novel endotype of asthma that is characterized by the dominance of dual positive Th2/Th17 cells in the airways. This Th2/Th17 predominant endotype distinguishes itself from the Th2 predominant and Th2/Th17 low endotypes with more severe asthma and greater refractoriness to treatment including steroids. The objective of this proposal is to study this Th2/Th17 predominant endotype of severe refractory asthma. We propose 4 specific aims. Under specific aim 1 we will characterize dual positive Th2/Th17 cells in airways from asthmatic patients and define the phenotype of Th2/Th17 predominant asthma. We will perform bronchoalveolar lavage, endobronchial biopsy and brushing in refractory asthmatic patients. We will characterize the cytokine and surface marker profile of BAL Th2/Th17 cells by flow cytometry and RNA-seq. Through analyses of tissue histopathologic, pulmonary physiologic, immunologic & clinical features we will identify unique phenotypic characteristics that are associated with the production of Th17 cytokines by Th2/Th17 cells. Specific aim 2 will examine the mechanism of development of airway Th2/Th17 cells in asthma. We will examine the role of IL1? and danger-associated molecular patterns in transdifferentiation of Th2 cells into Th2/Th17 cells. Under specific aim 3 we will delineate the signaling mechanism of steroid resistance of Th2/Th17 cells. The cytokines that induce Th2/Th17 cells also activate the MEK and p38 MAPK signaling pathways. We will examine the role of these signaling molecules in induction of steroid resistance. We will examine MEK regulation of the glucocorticoid receptor-associated co-repressor SMRT, p38 regulation of glucocorticoid receptor phosphorylation, and the consequences of these events for steroid resistance. Specific aim 4 will examine the role of IL1? and IL8 in the pathogenesis of neutrophilic asthma, another steroid resistant form of refractory asthma. We will also examine the role of Th2 cytokines in preventing neutrophil influx into the airways in Th2/Th17 predominant asthma. The study is important because it identifies and characterizes a novel endotype of severe refractory asthma. We will perform invasive procedures and examine bronchoalveolar lavage cells and bronchial tissue for vast majority of experiments, which has direct relevance for asthma. The delineation of the molecular mechanism of Th2/Th17 cell development and its steroid resistance will pave the way for clinical trials of already existing therapeutic agents in Th2/Th17 endotype of severe refractory asthma.